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Major urinary proteins (MUPs) are highly homologous proteoforms that function in binding, transporting and releasing pheromones in house mice. The main analytical challenge for studying variation in MUPs, even for state-of-the-art proteomics techniques, is their high degree of amino acid sequence homology. In this study we used unique peptides for proteoform-specific identification. We applied different search engines (ProteinPilot™vs. PEAKS®) and protein databases (MUP database vs. SwissProt + unreviewed MUPs), and found that proteoform identification is influenced by addressing background proteins (unregulated urinary proteins, non-MUPs) during the database search. High resolution Q-TOF mass spectrometry was used to identify and precisely quantify the regulation of MUP proteoforms in male mice that were reared in standard housing and then transferred to semi-natural enclosures (within-subject design). By using a designated MUP database we were able to distinguish 19 MUP proteoforms, with A2CEK6 (a Mup11 gene product) being the most abundant based on spectral intensities. We compared three different quantification strategies based on MS1- (from IDA and SWATH™ spectra) and MS2 (SWATH™) data, and the results of these methods were correlated. Furthermore, three data normalization methods were compared and we found that increased statistical significance of fold-changes can be achieved by normalization based on urinary protein concentrations. We show that male mice living in semi-natural enclosures significantly up-regulated some but not all MUPs (differential regulation), e.g., A2ANT6, a Mup6 gene product, was upregulated between 9-fold (MS1) and 13-fold (MS2) using the designated MUP database. Finally, we show that 85 ± 7% of total MS intensity can be attributed to MUP-derived peptides, which supports the assumption that MUPs are the primary proteins in mouse urine. Our results provide new tools for assessing qualitative and quantitative variation of MUPs and suggest that male mice regulate the expression of specific MUP proteoforms, depending upon social conditions.

Regulation of highly homologous major urinary proteins in house mice quantified with label-free proteomic methods